The present invention relates to a process for the preparation of modified hyaluronic acid (HA), in particular crosslinked HA in emulsion, for use in cosmetic and personal care applications.
Hyaluronic acid (HA) is a natural and linear carbohydrate polymer belonging to the class of the non-sulfated glycosaminoglycans. It is composed of beta-1,3-N-acetyl glucosamine and beta-1,4-glucuronic acid repeating disaccharide units with a molecular weight (MW) up to 6 MDa. HA is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis. HA may be extracted from natural tissues including the connective tissue of vertebrates, from the human umbilical cord and from cocks' combs. However, it is preferred today to prepare it by microbiological methods to minimize the potential risk of transferring infectious agents, and to increase product uniformity, quality and availability (WO 03/0175902, Novozymes).
Numerous roles of HA in the body have been identified. It plays an important role in the biological organism, as a mechanical support for the cells of many tissues, such as the skin, tendons, muscles and cartilage. HA is involved in key biological processes, such as the moistening of tissues, and lubrication. It is also suspected of having a role in numerous physiological functions, such as adhesion, development, cell motility, cancer, angiogenesis, and wound healing. Due to the unique physical and biological properties of HA (including viscoelasticity, biocompatibility, biodegradability), HA is employed in a wide range of current and developing applications within cosmetics, opthalmology, rheumatology, drug delivery, wound healing and tissue engineering. The use of HA in some of these applications is limited by the fact that HA is soluble in water at room temperature, i.e. about 20° C., it is rapidly degraded by hyaluronidase in the body, and it is difficult to process into biomaterials. Crosslinking of HA has therefore been introduced in order to improve the physical and mechanical properties of HA and its in vivo residence time.
U.S. Pat. No. 4,582,865 (Biomatrix Inc.) describes the preparation of crosslinked gels of HA, alone or mixed with other hydrophilic polymers, using divinyl sulfone (DVS) as the crosslinking agent. The preparation of a crosslinked HA or salt thereof using a polyfunctional epoxy compound is disclosed in EP 0 161 887 B1. Other bi- or poly-functional reagents that have been employed to crosslink HA through covalent linkages include formaldehyde (U.S. Pat. No. 4,713,448, Biomatrix Inc.), polyaziridine (WO 03/089476 A1, Genzyme Corp.), L-aminoacids or L-aminoesters (WO 2004/067575, Biosphere S.P.A.). Carbodiimides have also been reported for the crosslinking of HA (U.S. Pat. No. 5,017,229, Genzyme Corp.; U.S. Pat. No. 6,013,679, Anika Research, Inc). Total or partial crosslinked esters of HA with an aliphatic alcohol, and salts of such partial esters with inorganic or organic bases, are disclosed in U.S. Pat. No. 4,957,744. Crosslinking of HA chains with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDAC”) and adipic acid dihydrazide in a water/acetone mixture was disclosed in US 2006/0040892 (University of North Texas). WO 2006/56204 (Novozymes A/S) also discloses methods for the preparation of crosslinked gels of HA using divinyl sulfone (DVS) as the crosslinking agent.
WO 2008/100044 describes a method of preparing hyaluronic hydrogel nanoparticles by crosslinking hyaluronic acid, the method comprising mixing i) an oil phase containing a surfactant dissolved therein with ii) a water phase, containing hyaluronic acid and a water-soluble crosslinker, dissolved in an aqueous basic solution, so as to a form a w/o emulsion, and crosslinking the hyaluronic acid in the w/o emulsion, the oil phase comprising dodecane, heptane or cetylethylhexanoate.
EP 0 830 416 (equivalent of U.S. Pat. No. 6,214,331) describes the preparation of a crosslinked water-soluble polymer particle preparation wherein the particles are less than 212 μm in diameter and wherein at least 80% of the particles are spherical, obtainable by adding an aqueous polymer solution, comprising a water-soluble polymer selected from hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, celluloses, chitin, chitosan, agarose, carrageenans, curdlan, dextrans, emulsan, gellan, xanthans, poly(ethyleneoxide), poly(vinyl alcohol), poly(N-vinyl pyrrolidone), proteins, glycoproteins, peptidoglycans, proteoglycans, lipopolysaccharides, or combinations thereof, and an aqueous medium, to an oil base containing a water in oil emulsifying agent, agitating the mixture to form an emulsion containing polymer droplets, and crosslinking the polymer droplets in situ by a crosslinking agent resulting in the formation of crosslinked polymer particles. For the production of hyaluronic acid microspheres the crosslinking agent is added directly to an emulsion of aqueous hyaluronic acid in toluene. The crosslinking agent is first deactivated by adjusting the pH of the aqueous solution to pH 11 and then activated by lowering the pH to 7 to 8. It is preferred to use toluene, o-xylene or isooctane as oil phase. The weight ratio of aqueous phase to oil phase is about 1 to 1.
Nurettin Sahiner and Xinqiao Jai (Turk J Chem, 32 (2008), 397-409) describe the preparation of hyaluronic acid based submicron hydrogel particles using isooctane as oil phase. For preparing the emulsion 0.54 ml of aqueous hyaluronic acid solution was added to 15 ml of isooctane, resulting in a weight ratio of aqueous phase to oil phase is higher then 10 to 1.